Method of cultivating papaya plants and for recovering proteolytic enzymes from papaya plants



July 21, 1964 E. M. BURDICK METHOD OF CULTIVATING PAPAYA PLANTS AND FORRECOVERING Filed Jan. 7. 1960 PROTEOLYTIC ENZYMES FROM PAPAYA PLANTS 4[PAPAYA PLANTS}? 5 I q CHOP J: EXTRACTION PRESS I SOLUTION I a -F REwETPULP J PULPOUS ACIDIFYING -I PR SS MASS (E b AGENT 9 I III ENZYMATICDEHYDRATE DIGESTION Z PAPAYA PULP GREEN A COAc;ULUI\/I' I SEPARATION J--i PRECIPITATION OF PRECIPITATING /9-\ PROTEOLYTIC AGENTS ENZYMES z/ 1FINAL RECOVERY 22 x PURIFICATION OF PRECIPITATING YIN 4 DR I G AGENTS Z4PROTEOLYTIC ENZYMES F PURIFICATION J-vr Q FCONCENTRATION 1 IN VEN TOR.

BYW M United States Patent 3,141,832 METHGD @F CULTTVATING PAPAYA PLANTSAND FQR RECQVERHNG PRQTEGLYTEQ EN- ZYMES FRUM PAPAYA PLANTS Everette M.Eurdick, 4821 Ronda Se, Coral Gables, Fla. Filed Jan. 7, 1960, der. No.1,050 22 Claims. (Cl. 195-66) The present invention relates to a methodof cultivating papaya plants and to a process for recovering proteolyticenzymes from papaya plants.

The most important commercial product obtained from papaya is Papain.Papain acts in a manner similar to the better known animal pepsindigestive ferment, but it is superior to pepsin and other animalproteolytic enzymes in that it is effective over a wider pH range andresists higher temperatures before becoming inactivated. These uniqueproperties make papain the proteolytic enzyme of preference for manyindustrial applications such as, clarifying and stabilizing beer,tenderizing meats, hating hides, treating wool and silk textiles, cheesemanufacturing and in pharmaceuticals.

The papaya plant (Carica papaya) thrives throughout the tropics and canbe satisfactorily cultivated in certain parts of the United States suchas in Florida, Texas and California. All parts of the papaya plant havesupplied primitive people for a substantial period of time withmedicines, fibers, detergents, and so on, but it is primarily grown forthe papaya fruit which may be eaten. The mature fruit, which ismelonlike, supplies a fair share of the caloric, mineral and vitaminrequirements of tropical diets but by far the most important commercialproduct of the papaya fruit is papain which is simply the dried latex ofthe green immature papaya. fruit.

At the present time the general method of producing papain from greenfruit of the papaya plants is as follows. Papaya seeds are planted innurseries and then transplanted when about one month old, or when theyare 3 or 4 inches high. Often several seeds are planted directly in afield hole where the tree is wanted thereby eliminating transplanting,but it is necessary, when this procedure is followed to thin the plantsat a later date. The plants are normally spaced about 6 to 10 feetapart, which generally gives some 300 to 500 trees per acre.

When the sex of the trees can be determined definitely, they are thinnedso that a single tree is left in each hole, and it is desirable to leaveabout one male tree to each 30 or 50 female trees. ducing papain arenormally dioecious, that is, the male and female flowers are on separateplants. It is desirable, if not important, to have as many female orfruit producing trees as possible and yet have good pollination,

since it is the green papaya fruit that is tapped and from which thelatex is obtained. Weed cultivation may be practiced and fertilizer asnecessary must be applied to maintain satisfactory fruit and latexproduction. Commercial plantings may be subject to insect damage andvarious diseases which are ordinarily not found when papayas grow intheir native state or Wild, and these difiiculties must be overcome bythe use of expensive insecticides, fungicides, and cultural practices.

About eight months after transplanting, the papaya plants have producedimmature fruit which are ready for tapping; however, this time may varyconsiderably depending upon many factors. The fruit is tapped by makingcuts or incisions running the full length of the fruit, and the numberof cuts depends upon the size of the fruit, but generally consists offrom 2 to 5.

The number of incisions is not important, and some plantation operatorsprefer more or fewer incisions. Once the incisions are made the latexflows freely for only a few seconds and then coagulates. The latex mayThe best papaya plants for pro-,

be collected directly in a small container held by the tapper, but morecommonly it is allowed to drip onto an inverted umbrella or trayfastened around the trunk of the tree. From time to time the partiallydried and coagulated latex is scraped from the umbrellas and taken tothe drying shed where it is spread out on drying racks. These racks arethen placed in the kiln where heated air is made to circulate over themuntil the papain is thoroughly dried. When the drying operation isfinished the papain crum is then packed in tin containers.

Due to the economic factors involved, Africa and Ceylon are theprincipal sources of papain, and it can be observed that the manner ofcultivation and collection of the papain from the immature fruitinvolves the most primitive, and unsanitary methods which are directlyresponsible for many serious defects and limitations in papain which isavailable on the market today.

It is not uncommon for imported crude crum papain to be highlycontaminated with insects and other filth due to the above primitivemethod of collecting the papain, which makes most of the imported papainunsuitable for food and pharmaceutical use without further refining.

Also, papain offered on the market at the present time lacks uniformityin color, the color ranging from a dark brown to almost whitethe lighterthe color the better the grade in general, and enzymatic activity, whichis the most important property of the papain also lacks uniformity.

In addition, papain commercially available today likewise lacks goodsolubility, and is unstable on storage as well as possessing adisagreeable odor. The present invention relates to a new method ofcultivating papaya plants and for recovering the valuable proteolyticenzymes and other valuable constituents from the plant without thenecessity of producing papaya fruit.

The present invention provides a method of cultivating papaya wherebythe desirable constituents thereof may be recovered in a clean, sanitarymanner.

Another object of the present invention is to provide a method ofcultivating, harvesting and processing of immature green papaya plantsin order to obtain the valuable constituents from the plants without thenecessity of tapping the fruit of the papaya.

Yet a further object of the present invention is to provide a method ofprocessing the immature papaya leaves, stems and trunks for theproduction of papain.

Yet a further object of the present invention is to provide a process ofproducing papain having uniformity of color, uniformity of enzymaticactivity, and which possesses desirable odor as well as solubility andstability characteristics.

Another object of the present invention is to provide a method ofproducing a high grade sanitary papain that can be used directly infoods and pharmaceuticals.

Another object of the present invention is to provide a method ofproducing papain as Well as providing a source for other products suchas animal feedstufi's, chlorophylls, carotenes, xanthophylls, phytol,and carpaine.

The present invention relates to a new method of growing papaya plantsthickly planted and harvested mechanically while immature in a manner sothat the roots may put out new tops indefinitely, which immature papayaplants may thereafter be treated for extracting the valuable productstherefrom.

The present invention also relates to a process of recovering enzymesand other valuable constituents from immature papaya plants in a mannerwhich preserves, protects, activates and otherwise liberates thedesirable constituents from the plants.

Yet a further object of the present invention is to provide a processfor recovering proteolytic enzymes from immature papaya plants in amanner so as to protect the enzymes from certain natural enzymeinhibitors present, and more specifically, to a treatment of theimmature plants under controlled conditions of pH, time, andtemperature, so as to preserve, protect and activate the recoveredproteolytic enzymes.

Yet a further object of the present invention is to provide a method ofrecovering proteolytic enzymes from immature papaya plants therebyeliminating the necessity of obtaining the proteolytic enzymes from thegreen immature fruit of the plants.

Still a further object of the present invention is to recoverproteolytic enzymes from immature papaya plants, and to provide ananimal foodstulf from the processed plants which contains a relativelyhigh protein content and a relatively low crude fiber content.

Still a further object of the present invention is to provide a methodof recovering papain wherein the papain recovered is uniform in colorand is uniform in enzymatic activity as well as having desirablesolubility characteristics and which is stable over a long period ofstorage.

Other objects and advantages of the present invention will become moreapparent from a consideration of the following description and schematicflow sheet.

I have discovered that extremely large yields of a suitable source ofpapain may be obtained by planting papaya seeds at a rate of 10,000 to50,000 per acre. For example, the seeds may be planted every foot inrows 1 foot apart, thereby making it possible to obtain about 40,000plants per acre.

When the plants are about 2 to 3 feet tall, they are harvestedmechanically, as by a conventional forage harvester, and by cutting thegreen immature papaya plant trunks at a height of 9 to 12 inches abovethe ground, the roots will put out new tops rapidly.

Under good growing conditions, it is thus possible to make three or fourcuttings per year for several years before it becomes necessary toreplant the seeds. The present invention utilizes the green immatureplants as a source of desirable constituents, and it can be appreciatedthat the above procedure eliminates the time consuming and expensivetechniques heretofore used in cultivating papaya plants. Also, since thegreen immature papaya plants including the leaves, stems and trunks areused for the production of papain and other constituents, it can beappreciated that the problems involved in waiting on the plant toflower, and in tapping the plant used in present-day procedures areovercome.

As the plants are harvested it is advisable to spray the chopped plantswith a small quantity of preserving solution. The preserving solutionmay comprise a mixture of ammonia, sulphites and sulfides in the amountof 0.1% of each by weight. This concentration may vary without departingfrom the scope of the invention but is naturally restricted by economicfactors. Also, the pH of the spraying solution should be slightlyalkaline or neutral, and a pH range of 7 to 9 is quite satisfactory.

It is not necessary to spray the plants as they are harvested, but theyield of papain and the activity of the papain obtained from choppedplants when they are not sprayed may be reduced.

It should be further noted that while the present invention is describedin detail as being particularly applicable to the recovery ofconstituents from green immature papaya plants, it should be noted thatit may be profitable to process mature or older papaya plants that havebeen raised primarily for fruit. The process works equally well on olderplants; however, the young immature plants are preferred for severalreasons. First of all, they are more succulent, which makes them easierto chop and press in machinery both in the field and in the processingplant. Secondly, the enzyme yield per acre is generally far greater thanwhen older plants are used. Attention is now directed to the schematicflow diagram and it will be noted that the harvested plants representedby the numeral 2 after harvesting are chopped or macerated asillustrated at 3. After chopping the plants are treated with a smallquantity of extraction solution to horoughly Wet all the surfaces of theplants, especially the freshly cut or exposed surfaces to preventundesirable effects of oxidation and enzyme inactivation. This may bedone in any suitable manner and is most simply accomplished by sprayingthe chopped plants with the extraction solution; however, it may beaccomplished also by dipping the chopped pieces into the extractionsolution.

The extraction solution includes a chemical constituent or constituentswhich perform the function of a reducing or anti-oxidizing agent, apapain activator, and a solubilizing or peptizing agent. It is necessaryto provide a papain activator, and highly desirable to also have areducing agent and peptizing agent.

Among the reducing or anti-oxidizing agents which have been foundsuitable there are the sulfites and bisulfites of sodium and potassium,sulfur dioxide, the sulfides and hydrosulfides of ammonium, sodium andp0- tassium, hydrogen sulfide, glutathionine, methionine, cysteine,thioglycolates and cyanides. Out of this group, the sulfites andbisulfites of sodium, and hydrosulfides of ammonium and sodium arepreferred.

Among the papain-like enzyme activators which have been found suitable,there are the sulfites of sodium and potassium, the sulfides of sodiumand potassium, the bisulfites of sodium and potassium, hydrosulfides andsulfides of ammonium, sodium and potassium, certain metal chelatingagents (versene, sequestrene) like diethylenetetra amino acetic acid andits soluble salts, hydrocyanic acid and its sodium and potassium salts.Among this group, the sulfides of sodium, the bisulfites of sodium,hydrosulfides of ammonium and diethylenetetra amino acetic acid and itssoluble salts are preferred.

Among the solubilizing or peptizing agents, the following aredesirablechlorides of sodium and ammonium, phosphates of sodium andpotassium, various metaand polyphosphates of sodium and potassium,sodium citrate, hydrotropic agents such as sodium benzene sulfonate andsodium paracyrnene sulfonate. Among these, the chlorides of sodium andammonium, polyphosphates of sodium, and sodium citrate are preferred.Also, sodium sulfite, the bisulfides and sulfides of ammonium, andsodium, and ascorbic acid and its soluble salts are quite satisfactory.Generally speaking, any dilute salt solution will functionsatisfactorily.

Concentration of the chemicals may be in the neighborhood of 0.1% byweight, and concentrations in the range of 0.1% to 0.25% by weight aresatisfactory. Lower concentrations are inifective and higherconcentrations are not necessary to accomplish the results.

After the chopped mass of green papaya plants has been thoroughlymoistened, they are 'next pressed to separate a liquor or liquid fromthe pulpous mass of the papaya plants. The pressing may be accomplishedby any suitable apparatus, such as a filter press or the like toseparate the liquid from the pulp.

It is desirable to rewet the pulpous mass with the extraction solutionin order to extract more of the proteolytic enzymes and otherconstituents and this may be done by adding to the wet pulp a volume ofthe extraction solution about equal to the volume of liquid obtained inthe first pressing operation. The rewet pulp is then pressed as beforeto obtain a second portion of liquor and the resulting pulpous mass.

The pulpous mass may be dehydrated in a conventional dehydration kiln tomake papaya pulp which can be used as an animal feedstufi which containsa good amount of protein, and is quite rich in carotene or provitamin A.In the flow sheet the extraction solution is illustrated by the numeral4 and it can be seen that it is added as illustrated at 5 to the choppedpapaya plants and is added as illustrated at 6 to the rewet pulp.

After the pressing to separate the liquid from the pulp, the pressingsyield 75 to 90% of the total. The liquids are combined as illustrated at7 and 8 for subsequent processing. The pulpous mass obtained from thepress 9 is represented at 10 and as noted above is dehydrated at 11 toproduce the animal feedstuff represented by the numeral 12.

The two pressings of the plants yield 75% to 90% of the total enzymeactivity available from the plants and while more rewettings andpressings give higher yields of enzymes, the purification difficultiesof the enzymes later in the process appear to be increased.

Of course, the number of rewettings and pressings may be varied asdesired.

The combined liquors or liquids from the pressing may be green in colorand may have a pH in the neighborhood of 8. At any event, regardless ofthe color and regardless of the pH, the pH of the liquidis adjusted tobetween 4.0 and 4.5 by the addition of an acidifying agent asrepresented by the numeral 13 in the drawing. The acidifying agent maybe hydrochloric acid, sulfuric acid, citric acid, acetic acid, orphosphoric acid.

After the pH of the liquid has been adjusted, the temperature iscarefully raised to between 40 and 55 C. In this regard it should benoted that any localized overheating of the liquid will tend to destroythe proteolytic enzyme activity. Papain, like all enzymes, is destroyedby heat, but at temperatures below 55 C., the rate of destruction isquite low while at higher temperatures the rate of destruction increasesrapidly. The heating of the liquid effects enzymic digestion whichliberates and otherwise activates the proteolytic enzymes and at thesame time causes a coagulation of the protein impurities.

The enzymic digestion is continued until proper coagulation has beenobtained, and I have determined that the time required to produce thedesired results usually is between 10 and 30 minutes depending upon theparticular papaya plants from which the liquids were obtained. In thisregard, if the temperature of the liquid is raised carefully anduniformly, the coagulation will be effected at the lowest possibletemperature thereby producing the most active papain from the plants.Once the coagulate has been formed, the coagulate can be separated fromthe liquid by any suitable means such as filtration, centrifugation, orsimple settling. The formation of the coagulate may be termed a breakwhich is clearly visible to the eye in that the liquid seems to curdleand separate into coagulum which may be green in color and a clearsolution. Of course, any suitable instrumental means may be used todetermine the occurrence of this phenomenon.

Since the coagulate is mainly protein, it may be advisable to add somefilter aid at this point in order to effect more rapid filtration andoptimum clarification of the liquid. The clarified liquid can be furtherpurified by treatment with activated carbons and/or filter aids toproduce a relatively sparkling clear solution or liquid, which may thenbe concentrated from their 3 to solids content to some 30 or 50% solidscontent by means of vacuum concentration. In this regard, it should beagain noted that temperatures above 55 C. should be avoided to preventloss in enzymic activity.

In the flow diagram the enzymatic digestion is represented by thenumeral 14 and the separation at 15. The coagulate illustrated at 16 isa rich source of chlorophylls, carotenes, xanthophylls, carpaine, andprotein. If desired, the protein may be separated from the coagulate 16and added back to the papaya pulp feedstuif represented by the numeral12. The clear liquid separated from the coagulate is illustrated at 17as being purified as noted above, and concentrated as illustrated at 18and as noted hereinabove.

After the liquids have been purified and concentrated, the proteolyticenzymes may be separated therefrom by standard procedures. For example,the proteolytic enzymes can be precipitated by salting-out agents and bycertain water miscible organic solvents. The latter produces a betterproduct in that it is purer, while the former seems to be moreeconomical, and either may be used in the production of various gradesof proteolytic enzymes from papaya plants as necessary.

Precipitation of the proteolytic enzymes from the clarified concentratedsyrup may be most readily effected by means of ammonium sulfate. This ismost conveniently done by adding about two volumes of a saturatedammonium solution per each volume of concentration solution. Solidammonium sulfate can be used, the important thing being that a finalconcentration of 60% to 70% saturation, with respect to ammoniumsulfate, must be achieved. Lower concentrations of ammonium sulfate maybe used to purify further and to refine the enzymes present. Theprecipitated enzymes can be separated from the mother liquors by simplefiltration. If it is desirable to eliminate all traces of the bittertasting alkaloid carpaine, the precipitated enzymes should be washedwith a suitable organic solvent, such as toluene, before drying. Asnoted on the flow sheet, the enzymes are precipitated as represented at19 by the addition of precipitating agents 20, which precipitatingagents are recovered at 21 for reuse as noted in the flow sheet.

The ammonium sulfate can be recovered from the liquid for reuse as aprecipitating agent, or as a very valuable fertilizer.

Precipitation of the proteolytic enzymes from the clarified concentratedsyrup can also be effected by certain water miscible organic solventssuch as, methanol, ethanol, isopropanol, and acetone, which have allbeen found to be quite effective. In this regard, it should be notedthat it is necessary to add sutficient solvent to the concentratedenzyme solution to produce an organic solvent concentration of about 70%whereupon a fiocculent precipitate forms which may be separated, Washedwith fresh solvent and finally dried to produce high quality proteolyticenzymes. I prefer to use acetone since recovery for reuse is quitesimple by distillation. The use of water miscible organic solvents toprecipitate the proteolytic enzymes has another distinct advantage ofpermitting the recovery of the mother liquid syrup which is the richsource of the papaya alkaloid carpaine, and valuable nutrients which canbe added back to the papaya pulp feedstuif.

As noted at 22, the proteolytic enzymes may be subjeoted to the acetonepurification noted hereinabove if desired, and thereafter it may bedried by any suitable means as represented at 23 to produce theproteolytic enzyme product represented at 24.

To further amplify and describe the present invention the followingexamples are given:

Example 1.-A single young papaya plant about 2 feet tall was out about10 inches above the ground. The entire plant, consisting of trunk,leaves and stems, weighing 675 grams was chopped in simple food chopperfitted with a tapered screw. The pulpous mass thus produced, along withall expressed liquor, was placed in a cloth bag and then in a stainlesssteel fruit press provided with a hand operated screw to producepressure. About 300 milliliters of green press liquor was obtained. Thepulpous mass was rewet with 300 milliliters of extraction solutioncontaining 0.3 gram of sodium metabisulfite, 0.3

gram of tetrasodium diethylenetetraamino acetate, and 0.3 gram oftrisodium phosphate. After mixing in well the rewet mass was againpassed through the food chopper. The rewet mass was placed in the clothbag and pressed as before. Some 320 milliliters of green press liquorwas obtained. The pulpous mass was dried in an oven to make about gramsof papaya pulp, which contained some 14% crude protein and 182,000International Units of pro-vitamin A per pound. The green liquors werecombined and subjected to the following enzymatic digestion. Theoriginal pH of 7.9- was lowered to 4.2 by adding citric acid. Heat wasapplied to raise the temperature slowly from room temperature up to 35C., this took about 15 minutes and the solution was agitated slowly. Aslight break or separation of the green coagulum could be seen. Heatingand agitation were continued for about 20 minutes more, by which timethe temperature had reached 45 C. and the green coagulum had clearlyseparated. Filter aid was added and the green coagulum was recovered byfiltration. A straw colored filtrate was obtained, which was furtherpurified by treating with activated carbon and again filtered. Thealmost colorless purified solution was concentrated in a vacuum of 27inches of mercury to a thin syrupy consistency. The volume was estimatedat about 40 milliliters. This concentrate was treated with 200milliliters of acetone to precipitate the proteolytic enzymes as afiocculent white mass. The proteolytic enzymes were separated by suctionfiltration and washed wtih a small quantity of acetone. Vacuum dryingproduced 1.08 grams of material which possessed 440 milk-clotting unitsof enzymatic activity.

Example 2.The top was cut out of a mature papaya tree and portions ofthe leaves, stems, and trunk were chopped in a food chopper. Twokilograms of the macerated pulp plus drippings were placed in a clothbag and pressed in a stainless steel fruit press. Approximately 780milliliters of green press liquid were obtained having a pH very closeto 6. A One liter of extraction solution containing 1 gram of sodiumbisulfite, 4.5 milliliters of ammonium sulfide solution (22% (NH S), andenough sodium hydroxide to adjust the pH to 8.5 was used to rewet andthus extract the pressed pulp. The mass was thoroughly mixed and thenpassed through the food chopper. Upon pressing, almost 1200 millilitersof green press liquor was obtained. A portion of the pulp was dried inan oven maintained at 103 C. to produce a sample of papaya pulp of goodgreen color, which was assayed to contain 102% crude protein. The presscake was found to contain 68.2% moisture. The two portions of greenpress liquors were combined for the heat and enzymatic digestion tosolubilize and activate the proteolytic enzymes and to clarify thesolution. The pH of the combined green liquors was 8.0 and was adjusteddownward to 4.0 by adding dilute sulfuric acid. The solution wasmechanically agitated and heat applied to slowly raise the temperatureto about 40 C. where it was maintained for 1 hour. Some evidence of theprotein break was observed after about minutes, and by the end of thedigestion the green coagulum was well formed. It was removed by adding asmall amount of filter aid and filtering with suction, and the filtercake washed with a small quantity of distilled water. The filtrate wasyellowish orange, and had a volume of 2440 milliliters. This wasconcentrated in vacuum to about 400 milliliters and 2 liters of acetoneadded to precipitate the proteolytic enzymes as a light tan coloredfiocculent mass. The proteolytic enzymes were separated by filtrationand dried in vacuum. The material was tan colored, but grinding produceda light colored product. The yield was 2.81 grams which were found tohave 385 milk-clot ting units of enzyme activity per gram.

Example 3.-The top of young papaya plant weighing 725 grams was cut intopieces small enough to immerse in a portion of extraction solutioncontaining 0.1% sodium bisulfide, 0.1% potassium thiocyanate. Afterdipping in the extraction solution, the excess solution was allowedto'drain off and the pieces then chopped and macerated in the foodchopper. The pulpous mass along with all expressed juices were placed ina cloth bag and pressed to obtain 370 milliliters of green liquor. Thepressed pulp was rewet with 400 milliliters of the above extractionsolution and after thoroughly mixing and kneading it was again pressedto obtain 40 milliliters of green liquor. About 325 grams of pressedpulp was placed in an oven and dried at 103 C., to produce about 220grams of papaya pulp which assayed to contain 1.25% oil solublechlorophyll and 170,000 International Units of provitamin A. It was alsofound to contain 2.5% nitrogen or over 15% crude protein, using theconventional conversion factor of 6.25. The green liquors were combinedand the enzymic digestion conducted. The pH was adjusted to 4.0 byadding dilute hydrochloric acid. Agitation was started and the mixturewas heated to 38 C. in about 10 minutes, at which time the break couldbe seen. The heating was continued until the temperature reached 50 C.and maintained at this temperature for about 30 minutes, by which timethe green coagulum was well formed. The green coagulum was separated bycentrifugation and a yellowish colored clear centrifugate obtained. Asmall quantity of activated carbon was added and the solution maintainedat 50 C. with agitation for 10 minutes. The activated carbon was removedby suction filtration, and a light straw colored solution collected. Theclarified and purified proteolytic enzyme containing solution wasconcentrated under vacuum (27 in. Hg) to a syrupy consistency. Itsvolume was estimated at about 150 milliliters and 750 milliliters ofethanol added to produce a light tan fiocculent precipitate. This wasallowed to settle for some time and most of the mother liquor wasdecanted. The precipitated proteolytic enzymes were mixed with 150milliliters of fresh ethanol and separated by suction filtration andfinally washed with a small portion of absolute ethanol. Final drying invacuum produced 1.22 grams of light tan material that was found to 420milkclotting units of enzyme activity.

Example 4.-Two full grown papaya leaves weighing 260 grams were choppedand pressed to obtain 40 milliliters of green liquor. The pressed pulpwas rewet and thoroughly mixed with milliliters of extraction solutioncontaining 0.15 gram ammonium sulfide and 0.1 gram sodium chloride andagain chopped and pressed. This time milliliters of green liquor wereobtained, whose pH was 7.9. The pressed pulp weighing 129 grams wasdried to yield about 35 grams of deep green colored papaya pulp.Chemical assay showed that it contained about 2% chlorophyll. It was agood source of carotene since it was found to contain in excess of200,000 LU. units of provitamin A per pound. The crude protein contentwas about 30% and the crude fiber was about 5%. The green liquors werecombined and digested as follows. The pH was lowered to 4.2 by addingdilute hydrochloric acid and the temperature raised to 30 C. Mildagitation was used. A definite break could be seen after about 5minutes, and the heating and stirring were continued until a temperatureof 40 C. was reached. This took about 10 minutes and a good break hadbeen effected. Temperature was maintained at this point for another 30minutes. Filter aid and activated carbon were added and stirringcontinued for another 10 minutes, then filtered with suction. A lightclear yellowish-orange colored filtrate was obtained, which was reducedto about 20 milliliters under reduced pressure (about 27 in. Hg). Tothis thin syrup was added about 40 milliliters of a saturated solutionof ammonium sulfate to produce a precipitate. This solution was cooledabout 34 F. and let stand for several hours before the precipitatedproteolytic enzymes were separated by filtration. The precipitate wasfirst washed with a small quantity of ice water and then with absolutealcohol, before final drying. The recovered enyzrnes were tan colored,quite active in clotting milk, and weighed 0.43 gram. The green coagulumcontaining filter aid and carbon was found to be a suitable sourcechloroplyll, carotene, and xanthophyll.

Example 5 .-A single young papaya plant weighing 632 grams was choppedin a mechanical chopper fitted with a tapered screw and cutter. Themacerated mass was placed in a cloth bag along with the small quantityof expressed plant juices, and then pressed in a screw press to yield324 milliliters of green liquor. The enzymatic activity of thisexpressed green liquor was found to be very low as assayed by themilk-clotting test. The pulp was rewet with an extraction solutioncontaining 0.1% ammonium sulfide and 0.1% sodium chlorine by using 400milliliters. After thoroughly mixing, the mass was again passed throughthe chopper and mechanically pressed to obtain about 420 milliliters ofgreen liquor. A sample of this solution was assayed to have about twicethe milk-clotting activity as the first green liquor obtained. The greenliquors were combined and subjected to an enzymatic digestion to furtherfree and activate the proteolytic enzymes. The pH was raised to 8.5through the use of dilute sodium hydroxide, agitation was started andthe temperature raised to 35 C., at which time the pH was lowered to 4.0by adding dilute hydrochloric acid. These operations required about 15minutes. The temperature was then raised to 40 C. and the first signs ofthe separation or break were observed. The heating was continued foranother minutes, by which time the temperature had gone up to 50 C. Thetemperature was maintained at this temperature for some 25 minutes, bywhich time a good break could be seen clearly. The solution wascentrifuged to separate the green coagulum and a light orange solutionwas obtained. The green coagulum was rich in chloroplasts and a goodgrade of oil soluble chlorophyll could be extracted. It also was foundto be a satisfactory source of the papaya alkaloid carpaine (thedescription of the process employed for its isolation will be given inan additional disclosure). Approximately 7 grams of green coagulum wereobtained upon drying. The clarified centrifugate was then treated withgrams of activated carbon for about 10 minutes to remove moreimpurities, especially the color was improved. The clarified andpurified solution was tested for milk clotting activity and found to bevery active. The solution was concentrated to a syrup by means of vacuumevaporation. Its volume was estimated and five volumes of isopropanolwere added to effect precipitation of the proteolytic enzymes. Thismixture was placed in the refrigerator over night and the clearsupernatant liquor decanted. The flocculent somewhat gummy precipitatewas separated on a suction filter and washed with cold isopropanol, andthen dried. A yield of 0.45 gram of light colored material was obtainedhaving good milk-clotting proteolytic enzymic activity.

Example 6.Two small papaya plants that had been dipped in a watersolution containing 0.1% sodium sulfite for preservation were weighedsome 90 minutes later and found to weigh 838 grams. These were choppedin the mechanical chopper-grinder and the macerated mass along with theexpressed plant juices were mixed with 2 liters of water containing 0.1%sodium sulfite and 0.2% sodium polyphosphate and the mixture agitatedfor a few minutes, during which time the pH was found to be 7.1 whichwas raised to 8 through the addition of dilute ammonium hydroxide.Hydrogen sulfide was then passed through the agitated mixture untilsaturated and held for some or 30 minutes. The mixture was thentransferred to a cloth bag and mechanically pressed to obtain 1,410milliliters of green press liquor. This solution was found to be quiteactive in clotting milk, but it was further activated and clarified byfurther enzymatic digestion. The pH was lowered to 4.25 by adding dilutephosphoric acid and the temperature increased to 40 C. to eflect thefirst signs of a break or separation. Heating and stirring werecontinued for another 10 minutes to reach 55 C. and maintained there foranother 20 minutes. About 20 grams of filter aid and 20 grams ofactivated carbon were added and the heat treatment continued for 20minutes. Filtration produced a light tan clear filtrate which wasconcentrated under vacuum to a thin syrup. Addition of 5 volumes ofacetone produced an almost white fiocculent precipitate which wasseparated by suction. Contact of the precipitated proteolytic enzymeswith moist warm air at this point caused a darkening in color andsomewhat 10 of a gummy appearance. Treatment with absolute ethanolremoved the gumminess. Upon drying 0.67 gram of light tan coloredmaterial was recovered and assayed to have a high proteolytic activityas measured by the milk-clotting test.

Example 7 .-Several young papaya plants between 1 /2 and 3 feet tallwere out about 10 inches above the ground level. Within two weeks eachcorm (corms are the roots plus the trunk remaining after cutting off allstems, branches, leaves, etc.) had put out new tops that later formedtrunks, stems and leaves. Some corms put out more than one new top andthese were cut off in most cases. As many as twelve new tops have thusbeen obtained from a single root or corm. These plants were crushed andchopped in a stainless steel food chopper to form a thoroughly maceratedmass. Eight hundred grams of this mass were placed in a press and enoughpressure applied to express 432 milliliters of a green colored liquorwhich was found to contain 8.1% suspended and dissolved solids. Theproteolytic enzyme activity as measured by the standard milk-clottingtest was quite low. The green liquor was subjected to an enzymaticdigestion to liberate and otherwise activate the proteolytic enzymes,and at the same time to clarify and purify the solution to effect asatisfactory recovery of desired enzymes. The pH of the original greenliquor was raised from its value of 5.9 to 8.5 by adding dilute sodiumhydroxide. Then 0.5 gram of sodium sulfite and 0.5 gram of tetrasodiumdiethylenetetraamino acetate were added and the mixture stirred for 10minutes. The pH was then lowered to 4.4 by adding a dilute solution ofacetic acid. Agitation and heating were started, and the temperatureslowly raised to 40 C. where it was held until a visible separation ofthe green coagulum could be seen. This required some 30 minutes. Thetemperature was increased to 50 C. where it was held for another 20minutes, at which time a good separation had been effected, and a testof the mixture showed its milk-clotting activity had increased severalfold. The solution was centrifuged to separate the green curdled mass,or coagulum, which upon drying was found to weigh 8.9 grams. Subsequenttests showed it to be a good source of carpaine, protein, chlorophyll,carotene, and xanthophyll. The centrifugate was further purified bytreating with 20 grams of activated carbon, and 20 grams of filter cel,stirring for 10 minutes and then filtering. The purified and decolorizedsolution was concentrated under vacuum to a syrup and the proteolyticenzymes precipitated by adding five volumes of acetone. The flocculentprecipitate was recovered by filtration and the acetone saved forrecovery by simple distillation. Upon drying the precipitate it wasfound to weigh 0.64 gram. It was tan colored and fairly active in themilkclotting test.

What is claimed is:

1. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, treating the plants with an extractionsolution, pressing the treated plants to obtain a liquid from theplants, adjusting the pH of the liquid to 4.04.5, heating the liquid tobetween 40 -55 C. and digesting the heated liquid until a coagulate isformed therein, separating the liquid from the coagulate, purifying theliquid, concentrating the purified liquid, and treating the concentratedliquid to separate the proteolytic enzymes therefrom.

2. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, chopping the papaya plants, wetting thesurfaces of the chopped plants with an extraction solution, pressing thewetted plants to obtain a liquid from the plants, rewetting theresulting plant pulp with additional extraction solution, pressing therewet pulp to obtain additional liquid, combining the liquids from thepressing operations, dehydrating the pulpous plant mass, adjusting thepH of the combined liquids to between 4.04.5, heating the liquids tobetween 40-55 C. and digesting the heated liquids to form a menses i icoagulate therein, clarifying the liquids by separating the coagulatfrom the liquids, purifying the liquid, concentrating the liquid, andseparating the proteolytic enzymes therefrom.

3. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, treating the plants with an extractionsolution, pressing the treated plants to obtain a liquid from theplants, adjusting the pH of the liquid to 4.04.5, heating the liquid tobetween 4055 C. and digesting the heated liquid until a coagulate isformed therein, separating the liquid from the coagulate, and treatingthe liquid to separate the proteolytic enzymes therefrom.

4. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, chopping the papaya plants, wetting thesurfaces of the chopped plants with an extraction solution, pressing thewetted plants to obtain a liquid from the plants, rewetting theresulting plant pulp with additional extraction solution, pressing therewet pulp to obtain additional liquid, combining the liquids from thepressing operations, dehydrating the pulpous plant mass, adjusting thepH of the combined liquids to between 4.0-4.5, heating the liquids tobetween 40-55 C. and digesting the heated liquids to form a coagulatetherein, and separating the proteolytic en- 'zymes therefrom.

5. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, treating the plants with an extraction solutionincluding a reducing agent, papain activator, and a solubilizing agent,pressing the treated plants to obtain a liquid from the plants,adjusting the pH of the liquid to 4.04.5, heating the liquid to between4055 C. and digesting the heated liquid until a coagulate is formedtherein, separating the liquid from the coagulate, and treating theliquid to separate the proteolytic enzymes therefrom.

6. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, chopping the papaya plants, wetting thesurfaces of the chopped plants with an extraction solution including areducing agent, papain activator, and a solubilizing agent, pressing thewetted plants to obtain a liquid from the plants, rewetting theresulting plant pulp with additional extraction solution, pressing therewet pump to obtain additional liquid, combining the liquids from thepressing operations, dehydrating the pulpous plant mass, adjusting thepH of the combined liquids to between 4.0-4.5, heating the liquids tobetween 40-55 C. and digesting the heated liquids to form a coagulatetherein, and separating the proteolytic enzymes therefrom.

7. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, treating the plants with an extraction solutionincluding a reducing agent, papain activator, and a solubilizing agent,pressing the treated plants to obtain a liquid from the plants,adjusting the pH of the liquid to 4.0-4.5, heating the liquid to between40-55 C. and digesting the heated liquid until a coagulate is formedtherein, separating the liquid from the coagulate, purifying the liquid,concentrating the purified liquid, and treating the concentrated liquidto separate the proteolytic enzymes therefrom.

8. A method of treating papaya plants to recover proteolytic enzymescomprising the steps of, chopping the papaya plants, wetting thesurfaces of the chopped plants with an extraction solution including areducing agent, papain activator, and a solubilizing agent, pressing thewetted plants to obtain a liquid from the plants, rewetting theresulting plant pulp with additional extraction solution, pressing therewet pulp to obtain additional liquid, combining the liquids from thepressing operations, dehydrating the pulpous plant mass, adjusting thepH of the combined liquids to between 4.0-4.5, heating the liquids tobetween 40-55 C. and digesting the heated liquids to form a coagulatetherein, clarifying the liquids by separating the coagulate from theliquids, purifying the liquid,

i2 concentrating the liquid, and separating the proteolytic enzymestherefrom.

9. The process of claim 5 wherein the extraction solution reducing agentis taken from any of the following: the sulfites and bisulfites ofsodium and potassium, sulfur dioxide, sulfides and hydrosulfides ofammonium, sodium, and potassium, hydrogen sulfide, glutathionine,methionine, cysteine, thioglycolates, and cyanides, and wherein thepapain activator is taken from any of the following: sulfites of sodiumand potassium, sulfides of sodium and potassium, bisulfites of sodiumand potassium, hydrosulfides of ammonium, sodium and potassium,diethylenetetra amino acetic acid and its soluble salts, hydrocyanicacid and its sodium and potassium salts, and wherein the solubilizingagent is taken from any of the following: chlorides of sodium andammonium, phosphates of sodium and ammonium, meta and polyphosphates ofsodium and potassium, sodium citrate, and hydrotropic agents includingsodium benzene sulfonate and sodium paracymene sulfonate.

10. The process of claim 6 wherein the extraction solution reducingagent is taken from any of the following: the sulfites and bisulfites ofsodium and potassium, sulfur dioxide, sulfides and hydrosulfides ofammonium, sodium, and potassium, hydrogen sulfide, glutathionine,methionine, cysteine, thioglycolates, and cyanides, and wherein thepapain activator is taken from any of the following: sulfites of sodiumand potassium, sulfides of sodium and potassium, bisulfites of sodiumand potassium, hydrosulfides of ammonium, sodium and potassium,diethylenetetra amino acetic acid and its soluble salts, hydrocyanicacid and its sodium and potassium salts, and wherein the solubilizingagent is taken from any of the following: chlorides of sodium andammonium, phosphates of sodium and ammonium, meta and polyphosphates ofsodium and potassium, sodium citrate, and hydrotropic agents includingsodium benzene sulfonate and sodium paracymene sulfonate.

11. The process of claim 1 wherein the pH of the liquid is adjusted bythe addition of any of the following: hydrochloric acid, sulfuric acid,citric acid, acetic acid and phosphoric acid.

12. The process of claim 2 wherein the pH of the liquid is adjusted bythe addition of any of the following: hydrochloric acid, sulfuric acid,citric acid, acetic acid and phosphoric acid.

13. The process of claim 3 wherein the pH of the liquid is adjusted bythe addition of any of the following: hydrochloric acid, sulfuric acid,citric acid, acetic acid and phosphoric acid.

14. The process of claim 4 wherein the pH of the liquid is adjusted bythe addition of any of the following: hydrochloric acid, sulfuric acid,citric acid, acetic acid and phosphoric acid.

15. The process of claim 5 wherein the pH of the liquid is adjusted bythe addition of any of the following: hydrochloric acid, sulfuric acid,citric acid, acetic acid and phosphoric acid. 16. The process of claim 6wherein the pH of the liquid is adjusted by the addition of any of thefollowing: hydrochloric acid, sulfuric acid, citric acid, acetic acidand phosphoric acid.

17. The process of claim 7 wherein the pH of the liquid is adjusted bythe addition of any of the following: hydrochloric acid, sulfuric acid,citric acid, acetic acid and phosphoric acid.

' 18. The process of claim 8 wherein the pH of the liquid is adjusted bythe addition of any of the following: hydrochloric acid, sulfuric acid,citric acid, acetic acid and phosphoric acid.

19. The process of claim 5 wherein the extraction solution reducingagent is taken from any of the following: the sulfites and bisulfites ofsodium and potassium, sulfur dioxide, sulfides and hydrosulfides ofammonium, sodium, and potassium, hydrogen sulfide, glutathionine,methionine, cysteine, thioglycolates, and cyanides, and wherein thepapain activator is taken from any of the following: sulfites of sodiumand potassium, sulfides of sodium and potassium, bisulfites of sodiumand potassium, hydrosulfides of ammonium, sodium and potassium,diethylenetetra amino acetic acid and its soluble salts, hydrocyanicacid and its sodium and potassium salts, wherein the solubilizing agentis taken from any of the following: chlorides of sodium and ammonium,phosphates of sodium and ammonium, meta and polyphosphates of sodium andpotassium, sodium citrate, and hydrotropic agents including sodiumbenzene sulfonate and sodium paracymene sulfonate, and wherein the pH ofthe liquid is adjusted by the addition of any of the following:hydrochloric acid, sulfuric acid, citric acid, acetic acid andphosphoric acid.

20. The process of claim 6 wherein the extraction solution reducingagent is taken from any of the following: the sulfites and bisulfites ofsodium and potassium, sulfur dioxide, sulfides and hydrosulfides ofammonium, sodium, and potassium, hydrogen sulfide, glutathionine,methionine, cysteine, thioglycolates, and cyanides, and wherein thepapain activator is taken from any of the following: sulfites of sodiumand potassium, sulfides of sodium and potassium, bisulfites of sodiumand potassium, hydrosulfides of ammonium, sodium and potassium,diethylenetetra amino acetic acid and its soluble salts, hydrocyanicacid and its sodium and potassium salts, wherein the solubilizing agentis taken from any of the following: chlorides of sodium and ammonium,phosphates of sodium and ammonium, meta and polyphosphates of sodium andpotassium, sodium citrate, and hydrotropic agents including sodiumbenzene sulfonate and sodium paracymene sulfonate, and wherein the pH ofthe liquid is adjusted by the addition of any of the following:hydrochloric acid, sulfuric acid, citric acid, acetic acid andphosphoric acid.

21. A process of treating papaya plants to extract proteolytic enzymestherefrom including, wetting the plants with an extraction solution,pressing the wetted plants to separate the liquid from the pulp,adjusting the pH of the liquid to between 4.04.5, forming a coagulate inthe liquid by heating it to between 55 C. and digesting until thecoagulate forms, separating the coagulate, and treating the remainingliquid to separate the proteolytic enzymes therefrom.

22. A method of recovering a coagulate from papaya plants whichcoagulate is a rich source of chlorophylls, carotenes, xanthophylls,carpaine and protein comprising the steps of, treating the plants withan extraction solution, pressing the treated plants to obtain a liquidfrom the plants, adjusting the pH of the liquid to 4.0-4.5, heating theliquid to between 4055 C. and digesting the heated liquid until acoagulate is formed therein, and separating the coagulate and liquid.

References Cited in the file of this patent UNITED STATES PATENTS424,357 Russell Mar. 25, 1890 454,575 Weber June 23, 1891 1,959,750 WadaMay 22, 1934 2,227,818 Bayouth Jan. 7, 1941 2,257,218 Balls et al. Sept.30, 1941 2,313,875 Jansen 'et al Mar. 16, 1943 2,909,869 Dresser Oct.27, 1959 2,958,632 Schwarz et al. Nov. 1, 1960 OTHER REFERENCESChemurgic Digest, vol. 16, No. 7 (July 1957), pp. 4 to 6 and 12.

3. A METHOD OF TREATING PAPAYA PLANTS TO RECOVER PROTEOLYTIC ENZYMESCOMPRISING THE STEPS OF, TREATING THE PLANTS WITH AN EXTRACTIONSOLUTION, PRESSING THE TREATED PLANTS TO OBTAIN A LIQUID FROM THEPLANTS, ADJUSTING THE PH OF THE LIQUID OF 4.0-4.5, HEATING THE LIQUID TOBETWEEN 40*-55*C. AND DIGESTING THE HEATED LIQUID UNTIL A COAGULATE ISFORMED THEREIN, SEPARATING THE LIQUID FROM THE COAGULATE, AND TREATINGTHE LIQUID TO SEPARATE THE PROTEOLYTIC ENZYMES THEREFROM.